[unreadable] HIV infection and AIDS continue to be worldwide problems. In the United States alone, there are over 30,000 new cases of HIV infection per year. The advent of combination drug therapy or HAART has greatly changed the course of progression to AIDS for infected individuals, yet there are problems associated with the use of these drugs for many patients. These include difficult dosing regimens due to pharmacokinetic differences among individuals, disfiguring side effects, and the emergence of multi-drug resistant variants. The use of HAART is also lifelong and a major medical expense. It is therefore important that alternate approaches for the treatment of HIV infection be developed and tested. One such approach is the use of RNA interference, or RNAi. RNAi describes a complex set of post-transcriptional gene regulatory mechanisms triggered by short RNA duplexes. These mechanisms include gene silencing via sequence specific translational repression, cleavage of mRNAs and triggering of transcriptional silencing via sequence specific heterochromatin formation. In plants, worms and fruit flies RNAi is an anti-viral defense mechanism. Although it has never formally been proven to be a front line antiviral defense in mammals, all of the RNAi cellular machinery for anti-viral defense is present and readily programmable for viral inhibition when supplied with small RNA triggers. The proposed research capitalizes upon findings from the previous finding period which established principles and approaches for triggering anti-HIV RNAi in human hematopoietic cells. To date, ectopically triggered RNAi is one of the most powerful anti-HIV mechanisms known. Despite its potency, the fact that RNAi relies on Watson-Crick base pairing for target selection means that HIV can escape this inhibition via point mutations in the RNAi target regions. The best way to circumvent this is to use combinations of RNAi triggers, or small interfering RNAs (siRNAs) for the treatment of HIV infection via gene therapy of hematopoietic cells. Combinations of siRNAs triggering post-transcriptional gene silencing will be multiplexed with a strategy for using siRNAs to direct transcriptional gene silencing of the CCR5 co-receptor. The expression of these triggers will be carried out using novel, but well defined tRNA Pol III and U1 snRNA Pol II promoters. Various combinations of siRNAs and expression modes will be tested for safety and HIV efficacy in cell culture and then in a Rag2-/-?c-/- mouse model for human hematopoietic cell differentiation, maturation, and HIV challenge. The overriding hypothesis for these studies is that combinations of ectopically expressed siRNAs, targeting multiple sites and functions relating to HIV infection, can be used to circumvent viral escape mutants. The specific Aims of this study are as follows: 1) Mechanistic studies of siRNA selection and function in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS); 2) Exploring small RNA expression strategies for multiplexing RNAi PTGS and TGS triggers;and:3) Evaluate the stability, efficacy and potential toxicity of constitutively expressed shRNAs/miRNAs in CD34 cell derived macrophages and dendritic cells in vitro, and hematopoietic cells derived from in vivo humanized mice. The overall goal of this proposal is to develop potent and safe combinations of expressed siRNAs for the treatment of HIV infection in a hematopoietic cell gene therapy setting. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: HIV/AIDS continues to be a major threat to human health throughout the world. There is a continual need for new therapeutic approaches targeting HIV infection. RNA interference (RNAi) is a recently discovered powerful and naturally occurring mechanism for regulating gene expression. The significance of RNAi as a possible therapeutic is highlighted by the 2006 awarding of the Nobel Prize in Physiology and Medicine to the two scientists who discovered this phenomenon. This proposal is designed to exploit this natural process for the treatment of HIV infection by genetically engineering human blood cells to produce combinations of RNAi triggers called small interfering RNAs (siRNAs). These siRNAs will be rationally designed to provide maximal inhibition of HIV replication in the absence of toxicity. The primary goal of this research program is to validate RNA interference as a therapeutic approach for the treatment of HIV infection. [unreadable] [unreadable] [unreadable] [unreadable]